Citric acid derivatives

ABSTRACT

EPOXYACONITIC ACID AND ESTERS THEREOF ARE USEFUL FOR THE CONTROL OF LIPOGENESIS.

United States Patent 3,810,931 CITRIC ACID DERIVATIVES Robert WilliamGuthrie, Fairlield, James Guthrie Hamilton, Nutley, Richard WightmanKierstead, North Caldwell, and 0. Neal Miller, Montclair, N.J., and AnnClare Sullivan, New York, N.Y., assignors to Hoifmann-La Roche Inc.,Nutley, NJ. No Drawing. Filed Dec. 2, 1971, Ser. No. 204,334 Int. Cl.C07c 143/68; C07d 1/20, 1/22 U.S. Cl. 260-456 R 7 Claims ABSTRACT OF THEDISCLOSURE Epoxyaconitic acid and esters thereof are useful for thecontrol of lipogenesis.

BRIEF DESCRIPTION OF THE INVENTION The present invention relates tonovel epoxides of the formula ClICOzR C-CHzC 02R wherein all R groupsare either hydrogen or the same lower alkyl; and the stereoisomers,optical antipodes and pharmaceutically acceptable salts thereof, whichcompounds inhibit fatty acid synthesis in biological systems and arethus useful in the treatment of obesity and in correcting conditions oflipid abnormalities.

As used throughout the specification and the appended claims, the termlower alkyl shall mean a straight or branched chain hydrocarbon groupcontaining no unsaturation and having up to and including 8 carbonatoms, such as methyl, ethyl, hexyl, isopropyl, tert.-butyl and soforth. The term ary shall mean phenyl or naphthyl which may besubstituted with one of the following groups: halogen (i.e. chlorine,bromine, iodine or fluorine), lower alkyl, hydroxy, lower alkoxy ornitro.

The preparation of compounds of Formula I is illustrated in ReactionScheme A. The structural formulas depicted herein as drawn do notillustrate the relative or absolute stereochemistry of the particularmolecules. It should be understood, however, that all of the compoundsdescribed herein exist in two relative stereochemical forms: a cis andtrans form for compounds of formula II and a threo and erythro form forthe remainder of the compounds. To facilitate the description ofstereochemical transformations reported herein, the three and erythronomenclature as defined by Cram et al., J. Amer. Chem. Soc., vol. 74, p.5828 (1952) and by Prelog et al., Experientia, vol. 12, p. 81 (1956) hasbeen adopted. Each of these relative stereochemical forms exists as aracematc and as two optical antipodes, and the formulas shown herein aremeant to include all of the isomeric and antipodal forms of thecompounds depicted.

Epoxyaconitic acid, compound Ia, may be prepared by epoxidation ofaconitic acid. Trans-aconitic acid affords threo-epoxide andcis-aconitic acid affords erythroepoxide. The epoxidation is suitablyperformed utilizing an epoxidizing agent. Representative epoxidizingagents useful for this reaction include hydrogen peroxide and peracids.Suitable peracids include persulfuric acid, peracetic acid,trifluoroperacetic acid, mono perphthalic acid, perbenzoic acid,metachloroperbenzoic acid, and so forth. When hydrogen peroxide is usedas the epoxidizing agent, it is preferred to use an epoxidation catalystin conjunction therewith. A particularly effective epoxidation catalystis tungstic acid or a salt thereof, particularly an alkali metal salt.The epoxidation is preferably carried out in an aqueous medium, but anorganic solvent such as an alcohol or ether may be employed as a diluentif desired. Alternatively, the epoxidation may be performed in an inertorganic solvent such as a hydrocarbon, a halogenated hydrocarbon or anether. The reaction temperature is suitably in the range of from about 0C. to about 100 C. To avoid rearrangement of cis-aconitic acid totrans-aconitic acid prior to epoxidation, it is generally preferred toepoxidize the cis-acid between about 20 and Cis-aconitic acid anhydridemay be suitably employed in place of the acid.

REACTION SCHEME A CHCOzH CHCOiH OH -cn.co,rr o 00.11 02H (IIa) c-cmcom02H H030 CHzCOzH (In) on an ox cnoom' hnoom' o o-omoom' 4 no.0 cmoonz'01B. (Ib) H (V) V H00 n' =0 R'OrG CHzCOgR' --b q- H (IIb) more cmoouvH01 3 wherein -R' is lower alkyl and X is lower alkyl sulfonyl or arylsulfonyl.

The epoxidation of aconic esters of Formula 11b to epoxy esters ofFormula I'b may be accomplished in the same manner as described abovefor the conversion of 11a to Ia.

, Thevinterconversion of epoxides Ia and lb may be accomplishedaccordingto standard chemical procedures. For. example, the epoxy triacid offormula Ia may be esterified to epoxy triester of Formula Ib bycontacting said acid with a diazoalkylene such as diazomethane ordiazoethane according to well-known procedures. Conversely, the triesterof Formula Ib may be hydrolyzed to the triacid of formula Ia bytreatment with base. Suitable bases include for example alkali metalhydroxides, e.g. sodium hydroxide; and alkali metal carbonates orbicarbonates',.e.g. sodium carbonate or sodium bicarbonate. Thehydrolysis may be carried out in an aqueous alcoholic medium attemperatures ranging from about to about 50 C.

.It is generally not preferred to hydrolyze the erythroepoxy ester ofFormula Ib to the corresponding erythroepoxy acid of Formula Ia due tothe relative liability of the erythro-epoxides as compared with thecorresponding :threo-epoxides.

An alternative method for the preparation of epoxides of formula Ibinvolve, as a first step, cis-hydroxylation of aconitic ester 11b. Thecis-hydroxylation reaction may be accomplished by utilizing a peroxidein the presence of a hydroxylation catalyst. An especially preferredperoxide is hydrogen peroxide. An especially preferred hydroxylationcatalyst is osmium tetroxide. The catalyst may be employed in smallquantities as compared with the substrate to be hydroxylated, forexample from about 0.01 to about 25 mole percent. Approximately 0.1 molepercent of hydroxylation catalyst is preferred. The hydroxylation issuitably performed in any aqueous solvent or solvent mixture. Whenosmium tetroxide is utilized as a hydroxylation catalyst, it isgenerally preferred to treat the reaction mixture with a reducing agentbefore workup. Suitable reducing agents include sodium sulfite, sodiumbisulfite, sodium thiosulfate, and so forth. The reaction temperature ofthe hydroxylation reaction can be in the range from about 0 to about 100C. For example, if one utilizes. a trans-aconitic ester of Formula IIb,then a three-diol of formula IIIb is produced. The secondary hydroxylgroup of the diol of formula IIIb can then be selectively functionalizedto a suitable leaving group OX, wherein X is lower alkyl sulfonyl oraryl sulfonyl, by reaction with the appropriate sulfonyl halide.Suitable sulfonyl halides include methanesulfonyl chloride,p-toluenesulfonyl chloride, p-bromobenzenesulfonyl chloride,p-nitrobenzenesulfonyl chloride, and so forth. Methanesulfonyl chlorideis particularly preferred, in,-which case the compound of formula V is amethanesulfonate (mesylate) ester. The esterification is suitablyperformed in the presence of a base. Preferred bases are organic amines,e.g. pyridine, triethylamine and so forth. An excess of the amine canserve as the reaction solvent, or an inert organic solvent can serve asa diluent. Suitable diluents include hydrocarbons, e.g. benzene or.toluene; ethei's, e.g. ethyl ester or tetrahydrofuran; chlorinatedhydrocarbons, e.g. methylene chloride; and the like. The temperatureofthe esterifiction fraction can be in the. range from about to about 50C. A temperature range from about 0 to about 20 C. is preferred. In theesterification reaction there is no change in stereochemistry at thecarbon bearing the secondary hydroxyl. Thus, three-diol yieldsthreo-rnesylate and erythro-diol yields erythro-mesylate.

The sulfonate ester, compound V,,'is next converted to the epoxide offormula Ib by treatment with base. Suitable bases include, for example,salts of. lower alkanoic acids, for example sodium acetate or sodiumpropionate; alkali metal hydroxides, for example,

sodium hydroxide; alkali metal carbonates, for example, sodiumcarbonate; alkali metal hydrides, for example sodium hydride; and soforth. Choice of solvents and reaction temperatures are not critical andwill vary depending upon the nature of the base utilized. Suitablesolvents include lower alkanols, e.g. methanol or ethanol; hydrocarbons,e.g. benzene or toluene; and the like. When an alkanol is utilized assolvent, it is preferred to utilize the alkanol corresponding to the Rportion of the compound of formula V so that no trans-esterificationoccurs. When utilizing a base stronger than an alkoxide, it is preferredto use an aprotic solvent such as benzene or toluene so that reactionbetween the base and the solvent does not occur. Suitable combinationsof bases and solvents include, forexample, sodium hydride in benzene,sodium acetate in methanol, and so forth. The reaction temperature maybe in the range from about 20 to about 150 C. A lower reactiontemperature may be employed where a stronger base is utilized andviceversa. This elimination reaction takes place With inversion ofconfiguration. Thus, threo-mesylate affords erythro-epoxide anderythro-mesylate affords three-epoxide.

Epoxy triacids of formula Ia are readily converted to hydroxy citricacids of formula IIla which, in turn, form the corresponding -lactonesof formula IV. Compounds of formulas 111a and IV. are known usefulcompounds for the control of lipogenesis. See, for example, Belgian Pat.758,122. The conversion of Ia to IIIa may be accomplished by aqueouscleavage of the epoxide in the presence of either acid or base.Generally, the hydroxy citric acid IIIa is converted to the y-lactone IVin the reaction mixture during work up.

Suitable bases include alkali metal hydroxides, e.g. sodium hydroxide.As mentioned above, the hydrolysis reaction is carried out in an aqueousmedium. The reaction temperature can vary from about 20 to about 150.Generally, a reaction temperature of from about 50to about is preferred.

An epoxide of formula Ia may also be cleaved by heating alone in anaqueous medium; that is, the epoxy acid itself serves as the acidcatalyst. The reaction temperature for this autocatalyzed cleavage canbe in the range from about 20 to about although a temperature of fromabout 50 to about 100 is generally preferred. It is preferred to carryout the cleavage of the threo-epoxide under basic conditions and thecleavage of the erythro-epoxide. under acidic conditions.

In another variation, diol IIlb may be prepared directly from hydroxycitric acid 'y-lactone of formula IV by alkanolysis of the lactone ringand concomitant esterification of the carboxyl groups. In this reaction,the 'ylact0ne is treated with the desired alcohol, R'OH, wherein R is asabove, in the presence of an acid. In a preferred procedure, the alkanolserves as a reaction solvent, although any inert organic solvent can beutilized as a diluent. Suitable acids include mineral acids, e.g.hydrochloric acid or sulfuric acid; organic sulfonic acid, e.g.p-toluene sulfonic acid; and the like. The reaction temperature can bein the range from about 50 to about 100 C. A source of mineral acid canbe utilized in place of a mineral acid in this reaction, for example, anacid halide such as acetyl chloride can be added the reaction mixturecontaining the alcohol solvent to generate hydrogen chloride in situ.

Optically active compounds of formulas I, III and V may be prepared in anumber of ways. In one instance, compounds of formula Ia may be resolveddirectly. One resolution procedure involved fractional crystallizationof the salts of compound Ia with an optically active base, for example,an optically active amine. An especially preferred amine for effectingresolution of compounds of formula Ia is cinchonidine.

Alternatively, diol IIIb or sulfonate ester V can be resolved. Opticallyactive products may also be prepared starting with an optically activehydroxy citric acid of formula IIIa or its 'y-lactone of formula IV.

The compounds of formula I are useful for inhibiting fatty acidsynthesis in biological systems. The biological system in which thecompounds of the present invention may be used include those whichcontain citrate cleavage enzyme. Preferred biological systems aremammals, particularly non-ruminating mammals.

The inhibition of fatty acid synthesis in biological systems, by the useof the compounds of the present invention is believed to arise from theinhibition of citrate cleavage enzyme contained in such systems. Thecleavage of citrate is catalyzed by citrate cleavage enzyme according tothe stoichiometry:

citrate+CoA+ATP- acetyl-CoA +oxaloacetate +ATP+P In the conversion ofcarbohydrates and various amino acids to fat by non-ruminating mammals,citrate is the major source of acetyl co-enzyme A which is utilized forthe synthesis of fatty acids. Citrate is formed in the mitochondria bythe citrate synthase reaction. It is then metabolized via the citricacid cycle. Under conditions when energy intake exceeds energy demand,some citrate is diverted to the extra-mitochondrial space of the cellwhere it is used for fatty acid synthesis, that is to say, for energystorage. The novel compounds of formula I of the present invention arethus useful in the treatment of obesity and in the correction of lipidabnormalities. The epoxides of formula Ia may also be utilized in theform of their pharmaceutically acceptable non-t0xic basic salts.Preferred salts for this purpose include the alkali metals, e.g. sodiumor potassium; the alkaline earth metals, e.g. calcium; or complex saltssuch as ammonium or substituted ammonium salts such as a mono-, or diortri-alkylammoniurn salt or a mono-, dior tri-hydroxyalkylammonium salt.The compounds can 'be made up in the form of conventional pharmaceuticalpreparations; for example, the aforesaid compounds can be mixed withorganic or inorganic inert pharmaceutical carriers suitable forparenteral or enteral administration such as, for example, water,gelatin, lactose, starch, magnesium stearate, talc, vegetable oil, gumsor the like. They can be administered in conventional pharmaceuticalforms, e.g. solid forms, for example, tablets, capsules, drages,suppositories, or the like; or in liquid forms, for example, solution,suspensions, or emulsions. Moreover, the pharmaceutical compositionscontaining the compounds of this invention can be subjected to theconventional pharmaceutical expedients, such as sterilization, and cancontain conventional pharmaceutical excipients such as preservatives,stabilizing agents, emulsifying agents, salts for the adjustment ofosmotic pressure or buffers. The compositions can also contain othertherapeutically active materials.

\A suitable pharmaceutical dosage unit can contain from about to 600 mg.of the aforesaid compound.

Suitable parenteral dosage regimens in mammals comprise from about 1mg./kg. to about mg./kg. per day. However, for any particular subject,specific dosage regimens should be adjusted according to the individualneed and the professional judgment of the person administering orsupervising the administration of the aforesaid compounds. It is to beunderstood that the dosages set forth herein are exemplary only; thatthey do not, to any extent, limit the scope or practice of theinvention.

The present invention may be more clearly illustrated by the followingexamples. All the temperatures are stated in degrees centigrade.

EXAMPLE 1 i) -Threo-1,2epoxy-1,2,3-propanetricarboxylic acid i-threo-epoxyaconitic acid] A solution of trans-aconitic acid (10 g.) andtungstic acid (2.0 g.) in 1 N sodium hydroxide solution (143 ml.)containing hydrogen peroxide 6 ml.) was stirred at for two hours. Thesolution was cooled, 10 N by drochloric acid (15.5 ml.) was added andthen was extracted exhaustively with ether using a liquid-liquidextractor. Evaporation of the ether layer furnished a white solid whichon crystallization from ether-methylene chloride gave epoxide, MAP.167-169". A second crop, 159-164 was isolated from the mother liquors.The analytically pure specimen was obtained from the same solventsystem, M.P. 168170.

Analysis.Calcd. for CeHsOqI C, 37.91; H, 3.18. Found; C, 37.22; H, 3.69.

EXAMPLE 2 Resolution of (i -threo-1,2-epoxy-1,2,3-propanetricarboxylicacid (i -threo-epoxyaconitic acid] A solution of (:)-thre0-epoxyaconiticacid (3.0 g.) in methanol ml.) was heated to reflux. To the boilingsolution cinchonidine (9.0 g.) was added followed by ethyl acetate (-250ml.) and heating was continued until crystallization of the salt hadstarted. The solution was allowed to cool and the cinchonidine salt wascollected by filtration and washed with ethyl acetate (20 ml.) (Crop A).The filtrate was concentrated and two additional crops of crystallinematerial were obtained (Crops B and C). The mother liquors wereevaporated to dryness in vacuo to give a white solid (Crop D). Crops Aand D were processed as below. Crops B and C were combined forrecycling.

(i) (+)-'Ihreo-epoxyaconitic acid.Crop A was dispersed in chloroform andextracted with 1 N sodium hydroxide solution (1X 25 ml.; 1X 10 ml.). Thecombined basic extracts were washed with chloroform (2X 10 ml.) thenwere acidified with 1 N hydrochloric acid (36 ml.) and concentrated invacuo. The resulting residue was extracted with hot ethyl acetate andthe residual sodium chloride was removed by filtration. The filtrate wasconcentrated under reduced pressure and crystallization of the productfrom ethyl acetate-carbon tetrachloride gave the epoxide as a carbontetrachloride solvate. Air drying furnished the dextrarotatorythreo-epoxide as the monohydrate, M.P. 108l12; [u] +63.1 (c., 1.0, H 0).

Analysis-Calcd. for C HGOq'HzOI C, 34.63; H, 3.87. Found: C, 34.91; H,3.81.

(ii) ()-Threoepoxyaconitic acid.-Crop D was dispersed in chloroform (40ml.) and extracted with two portions (35 ml. and 5 ml.) of 1 N sodiumhydroxide solution. The combined aqueous extracts were washed withchloroform (2X 10 ml.) then were acidified with 1 N hydrochloric acid(51 ml.) and evaporated to dryness in vacuo. Trituration of the residuewith ethyl acetate and evaporation of the extracts gave an oil which onfractional crystallization from ethyl acetate-carbon tetrachloride gavethe solvated levorotatory epoxide. After air drying, the monohydrate hadM.P. 108-112; [M --62.5 (c., 1.0, H 0).

Analysis.Calcd. for C H O -H O: C, 34.63; H, 3.89. Found: C, 34.91; H,3.74.

EXAMPLE 3 (+)-Erythro-hydroxycitric acid, 1 lactone (Hibiscus acid) from(+)-threo-epoxyaconitic acid A solution of dextrarotatory epoxideprepared as in Example 2 (1.9 g.: 30 meq.) in 1 N sodium hydroxide (30ml.) was refluxed under nitrogen for three days. The reaction mixturewas then cooled to -80 and a solution of calcium chloride (2.6 g.) inwater (5 ml.) was added rapidly with stirring. The resulting whiteprecipitate was collected by filtration and washed well with water thenwas dissolved in the minimum amount of 1 N hydrochloric acid (-15 ml.).The solution was passed through a column of cationic exchange resin(Amberlite IRA 120; 30 ml.). The acidic eluents were evaporated todryness and then the residue was heated on a steam bath to inducecrystallization to give essentially pure lactone.

The residue was dissolved in ethyl acetate and the pale yellow solutionWas decolorized with charcoal. Two crystallizations from ethylacetate-carbon tetrachloride furnished analytically pure hibiscus acid,M.P. 187-8; [(11 +109" (0., 1.0, H 0), identical with authenticmaterial.

EXAMPLE 4 (1L)-Erythro-l,2-epoxy-1,2,3-propanetricarboxylic acid (-J:-erythro-epoxyaconitic acid A solution of tungstic acid (1.3 g.) inhydrogen peroxide 6.6 ml.) was added to cis-aconitic anhydride (10.1 g.)in water (115 ml.) and the stirred reaction mixture was placed in an oilbath previously heated to The reaction was maintained at thistemperature for 90 minutes then it was placed in a liquid-liquidextractor and continuously extracted with ether. Concentration of theextract obtained after 5.5 hours gave an oil. Fractional crystallization(ethyl acetate-carbon tetrachloride) of this material gave theerythro-epoxide, M.P. 176178 and a second crop, M.P. 168-172. Thematerial obtained from continuous extraction overnight furnishedadditional epoxide, M.P. 173176.

The pure (i-)-erythro-epoxide was obtained by crystallization from ethylacetate-carbon tetrachloride, M.P. 175.5-178".

Analysis.-Calcd. for C H O C, 37.91; H, 3.18. Found: C, 37.96; H, 3.44.

EXAMPLE 5 (:t)-Threo-hydroxycitric acid, 1 lactone from (i)-erythro-epoxy aconitic acid A solution of the erythro-epoxide preparedas in Example 4 (190 mg.) in water (3 g.) was heated on a steam bath for4.5 hours. The solvent was removed in vacuo and the oily residue washeated on a steam bath for 30 min. to give 190 mg. of(i)-threo-hydroxycitric acid 'y lactone. A small amount was esterifiedusing diazomethane in ether. Examination of the resulting ester showedit to be essentially pure (:)-thre0-hydroxycitric acid, 7 lactone,dimethy lester.

EXAMPLE 6 Trans-trimethyl aconitate Acetyl chloride (100 ml.) was addedto a cooled solution of trans-aconitic acid (100 g.) in methanol (1500ml.). The solution was refluxed for 3.5 hours then was cooled to roomtemperature. Sufficient pyridine (86 ml.) was added to neutralize thereaction mixture, then most of the solvent was removed in vacuo. Theresulting oil was dissolved in methylene chloride and the solution waswashed in turn with dilute hydrochloric acid solution, water, dilutesodium bicarbonate solution and finally water. The methylene chlorideextract was dried (MgSO and concentrated under reduced pressure to givethe trimethyl ester as a pale yellow oil. This material was distilledunder vacuum to afford the triester as a colorless liquid (B.P.110-112"; 0.6 mm.).

EXAMPLE 7 (i -Threo-l,2-dihydroxyl ,2,3-propanetricarboxylic acid,trimethyl ester To a solution of trans-trimethyl aconitate (57 g.) inacetone (300 ml.) and water (75 ml.) was added a solution of osmiumtetroxide in acetone (1%; 7.5 ml.) followed by hydrogen peroxide (30%;37 ml.). The stirred solution was brought to reflux and was maintainedat this temperature for four hours. The reaction mixture was cooled andleft at room temperature overnight, then enough sodium bisulfite wasadded (-1 g.) to discharge the remaining oxidant. The solvent wasremoved in vacuo and the residual oil was dissolved in water (300 ml.)and extracted with ether (4 250 ml.). The ether layers were backwashedin turn with water (100 ml.). Sodium chloride (130 g. was dissolved inthe combined aqueous layers which were then extracted with methylenechloride (5 X 300 ml.). The combined methylene chloride extracts weredried (MgSO and evaporated to give a viscous oil. Crystallization of theoil from ether-hexane afforded 29.3 g. of the (i-threo-diol, M.P.74-76". The analytical sample was obtained from the same solventmixture, M.P. 7576.

Analysis.Calcd. for C H O C, 43.20; H, 5.64. Found: C, 43.42; H, 5.68.

EXAMPLE 8 )-Threo-1-mesyloxy-2-hydroxy-1,2,3-propanetricarboxylic acid,trimethyl ester Methanesulfonyl chloride (4.5 ml.; 57 mmols) was addedto a cooled (-5) solution of the (i)-thre0-diol prepared as in Example 7(12.0 g.; 48 mmols) in pyridine ml.) and the mixture was kept at 0-5 for2 hours. Several chips of ice were then added to the reac tion mixtureand after five minutes it was poured into an ice-water mixture (500 ml.)containing concentrated hydrochloric acid ml.). The pale yellow acidicsolution was extracted with methylene chloride (3X 200 ml.) and theorganic layers were washed in turn with brine and with a saturatedsodium bicarbonate solution. The dried (MgSO methylene chloride extractswere decolorized (charcoal) and then evaporated to dryness.Crystallization of the residue from methylene chloridehexane gave paleyellow crystals, M.P. 92-94. The analytically pure mesylate was obtainedby crystallization from ethyl acetate-hexane, M.P. 91-93.

Analysis.Calcd. for C H O S: C, 36.56; H, 4.91; S, 9.76. Found: C,36.41; H, 4.95; S, 9.72.

EXAMPLE 9 i)-Erythro-1,2-epoxy 1,2,3 propanetricarboxylic acid,

trimethyl ester [(i -erythro-epoxyaconitic acid, trimethyl ester] Asolution of (i)-threo-mesylate prepared as in Example 8 (56.0 g.) inmethanol (800 ml.) containing sodium acetate (28.0 g.) was refluxed for100 minutes and then was cooled to room temperature. A copiousprecipitate that had formed during the reaction was removed byfiltration and discarded and the filtrate was concentrated in vacuo. Theresidue was taken up in chloroform and the solution was washed in turnwith brine and dilute sodium bicarbonate solution. The dried (MgSOorganic layer was evaporated under reduced pressure and the resultingoil was crystallized from ether-hexane to give the epoxide, M.P. 54-57.Analytically pure epoxide was obtained from ether-hexane, M.P. 55-57.

Analysis.Calcd. for C H O C, 46.56; H, 5.21. Found: C, 46.66; H, 5.16.

EXAMPLE 10 (i)-Threo-1,2-epoxy-1,2,3-propanetricarboxylic acid,trimethyl ester -)-threo-epoxyaconitic acid, trimethyl ester] Acetylchloride (20 ml.) was added carefully to a solution of(i)threo-epoxyaconitie acid (20 g.) in methanol (400 ml.) previouslycooled by means of an ice-water bath. The solution was refluxed for onehour, then was cooled and enough pyridine (25 ml.) was added toneutralize the reaction mixture. The solvent was removed in vacuo andthe resulting oil was partitioned using methylene chloride and water.The methylene chloride layer was washed with brine, 0.5 N hydrochloricacid solution and finally dilute sodium bicarbonate solution. Theaqueouslayers were backwashed in turn with methylene chloride, then thecombined organic extracts were dried (MgSO and concentrated underreduced pressure to give a viscous oil. Crystallization of the. crudeproduct from ether-hexane furnished essentially pure epoxide,

concentrated under 'reduced pressure to 9 M.P.. -34". The analyticalsample was obtained from the same solvent, M.P. 38-40.

Analysis.Calcd. for C H O r Found: C, 46.68; H, 5.03.

EXAMPLE 11 To a solution of (i)-erythro-hydroxycitric acid, 'y lactone;(21 g.) in methanol (400 ml.) was added acetyl chloride (21 g.). Thesolution was brought to reflux and maintained at this temperature for 90minutes, then was cooled and left overnight at room temperature. Enoughpyridine (19 ml.) was added to neutralize the reaction mixture then thesolvent was removed under reduced pressure. The resulting syrup wasdissolved in water (350 ml.) and the solution was extracted with ether(5X 100 ml.). The ether extracts were backwashed in turn with water (2X100 ml.). Sodium chloride (-125 g.) was dissolved in the combinedaqueous layers and the solution was extracted with methylene chloride(1x 350 ml.; 3x 125 ml.). The methylene chloride extracts were washed inturn with brine and with saturated sodium bicarbonate solution. Thedried (MgSO methylene chloride extracts were concentrated in vacuo togive the trimethyl ester as a viscous oilr EXAMPLE l2 1 (i-Erythro-l-mesyloxy-Z-hydroxy-1,2,3-propanetricarboxylic acid,-trimethylester Crude (1)-erythro-di0l (23.7 g.) prepared asin Example 11 wasdissolved in pyridine (200 ml.) and to the cooled (-5) solutionmethanesulfonyl chloride (7.2 ml.) was added. The reaction mixture wasstored at -5 for 150 minutes then it was poured into an ice-watermixture (-1 1.) containing concentrated hydrochloric acid (210 ml.). Theresulting red solution was extracted with methylene chloride (4x 200ml.)and then the organic layers were washed in turn with brine and withsaturated sodium bicarbonate solution. The combined methylene chlorideextracts were dried (MgSO then decolorized (charcoal) and concentratedunder reduced pressure to give a pale yellow oil'. Triturationof theresidue with ether furnished crude mesylate which on'recrystallizationfrom methylene chloride-hexane gave 21.7 g. of pure material, M.P.104-106.

Analysis.--Calcd. for C H O 'S: C, 36.58; H, 4.91; S, 9.76. Found: C,36.63; H, 4.85;S, 9.81. 1

EXAMPLE l3 -Threo-1,2dihydroxy-1,2,3-propanetricarboxy1ic acid,trimethyl ester To a cooled solution of (+)-thre0-hydroxycitric acid '7lactone (garcinia" acid; 10 g.) in methanol (200 ml.) was added acetylchloride (10 1111:). The solution wan heated at reflux for 90 minutesthen cooled toroom temperature. Suflicient pyridine (9.0 ml.) was addedto neutralize the reaction mixture then the solvent was removed invacuo. The oily residue was dispersed in brine and extracted withmethylene chloride (x 100 ml.). The organic extracts were washed in turnwith 1N hydrochloric acid solution and with-saturated sodium bicarbonatesolution. The extracts were combined, dried (MgSO and give 'crude dioltrimethyl ester asan oil.

EXAMPLE 14 I t 1 (S ,2 (S) 1-mesyloxy-2-hydroxy-1 ,2,3-propanetricarboxylic acid, trimethyl ester )-threo-rne sylate] Methane-sulfonylchloride (3.55 ml.) was added to a solution of crude ()-thre0-diolprepareda's in Example 13 (11.7 g.) in pyridine (100 ml.) previouslycooled to 5-10. The reaction mixture was kept at 0-5' for 2.5-

(.1) .All' the ingredients were mixed thoroughly and Dica c h sp a' ihydla't hours then was poured into a stirred ice-water mixture containingmls. of concentrated hydrochloric acid The resulting solution wasextracted with methylene chloride (4 '200 ml.) and the extracts Werewashed in turn with brine (1 X and saturated sodium bicarbonate (2X Thecombined extracts were dried (MgSO decolorized (charcoal) andconcentrated in vacuo to give the crude mesylate as an oil. Triturationof the oil with ether alforded a solid, M.P. 83-86". Recrystallizationof this material from methylene chloride-hexane gave the mesylate, M.P.89-91. The analytically pure sample was obtained by crystallization fromether-hexane, M.P. 89-91 8.38 (c., 0.75, CH OH).

Analysis.-Calcd. for C H O S: C, 36.58; H, 4.91; S, 9.76. Found: C,36.78; H, 4.97; S, 9.73.

EXAMPLE 15 A solution of the optically active mesylate prepared as inExample 14 (15 g.) in methanol (200 ml.) containing sodium acetate wasstirred under reflux for 2 hours. The reaction mixture was cooled andmost of the methanol was evaporatedunder reduced pressure. The residuewas taken up,.in chloroform and the chloroform solution was washed inturn with brine, sodium bicarbonate solution and finally brine. Theorgam'c layer Was dried and evaporated in vqcuo tq give an oil.Crystallization of the product from ether gave, the epoxide, M.P. 64-5.The same solvent furnishedthe analytically pure sample, M.P. 645; -34.6.(c. 1.07, CH OH).

.Anqlysix-Calcd. for CgHmOqI C, 46.56; H, 5.21. Found: C, 46.71; H,5.08.

EXAMPLE 16 Capsule formulation Per capsule, mg. (i)-Threo-epoxyaconiticacid 250 Lactose 60 Corn starch 35 Magnesium stearate 5 Total weight 350Procedure EXAMPLE- 17 v Procedure FitzedlModel 'D) usinga #IA screen,medium speed.

2) The mixture was remixed and 'slugged. (3) The"slugs were screened onan oscillator through a #14 mesh screen and compressed'on an E machine.

1 1 'EXAMPLE 1s Capsule formulation Per capsule, mg.(i)-Threo-epoxyaconitic acid 50 Lactose, U.S.P. 125 Corn starch, U.S.P30 Talc, U.S.P.

Total weight 210 Procedure EXAMPLE 19 Tablet formulation Per tablet, mg.

(-+ -)-Threo-epoxyaconitic acid Dicalcium phosphate dihydrate, unmilled175 Corn starch 24 Magnesium stearate 1 Total weight 225 Procedure (1)(i)-Threo-epoxyaconitic acid and corn starch were mixed together andpassed through a #00 screen in Model J Fitzmill with hammers forward.

(2) This premix was then mixed with dicalcium phosphate and one-half ofthe magnesium stearate, passed through a. #1A screen in Model J Fitzmillwith knives forward, and slugged.

(3) The slugs were passed through a #2A plate in a Model D Fitzmill atslow speed with knives forward, and the remaining magnesium stearate wasadded.

(4) The mixture was mixed and compressed.

EXAMPLE 20 Tablet formulation Per tablet, mg. (i)-Threo-epoxyaconiticacid 100 Lactose, U.S.P. 202 Corn starch, U.S.P. 80 Amijel B011 1 20Calcium stearate 8 Total weight 410 A prehydrolyzed food grade cornstarch, Any similar prehydrolyzed corn starch may be used.

Procedure 12 (4) The mixture was compressed at a tablet weight of 410mg. using tablet punches having a diameter of approximately (Tablets maybe either flat or biconvex and may be scored if desired.)

EXAMPLE 21 Tablet formulation Per tablet, mg.

(1)-Threo-epoxyaconitic acid 500 Corn starch 30 Lactose 88 Gelatin v 12Talcum l5 Magnesium stearate 5 Total weight 650 Procedure (1)(1)-Threo-epoxyaconitic acid and lactose were thoroughly mixed insuitable blending equipment and granulated with a 10% gelatin solution.

(2) The moist mass was passed through a #12 screen, and the granuleswere dried on paper lined trays overnight.

(3) The dried granules were passed through a #14.

screen and placed in a suitable mixer. The talcum and magnesium stearatewere added and blended.

(4) The granulation was compressed into tablets weighing approximately650 mg. each, using punches having an approxmate diameter of 12.7 mm.(/2"). The final tablet thickness was about 5.1 mm.

EXAMPLE 22 Measurement of lipogenesis in vivo Female Charles River ratsweighing from 120-150 g. were provided free access to water and were feda commercial diet prior to the initiation of the experiment. Eachexperimental group of animals were pro-fasted two days and then meal-feda single meal daily from 9-12 am. The meal consisted of a glucosefat-free diet (G-70) containing 70% glucose, 24% vitamin-free casein, 5%salt and 1% vitamin, to which 40 g. cellulose was added per kilogram.

On the last day of feeding, at a specified time before initiation of themeal, the epoxide derivative in ASV of the composition sodium chloride0.9%, carboxy methyl cellulose 0.5%, benzyl alcohol 0.86% 'and Tween(polyoxyethylene sorbitan monoleate) 0.39% or in saline solution wasadministered. At a specified time after feeding, rats were lightlyanaesthetized with Penthrane (methoxyflurane) and injected in the tailvein with 0.25 ml. of a solution with the following composition: 12.3mg. alanine, 5 c. C-alanine (specific activity= l56 mc./ mmole) as fattyacid precursor and 30.6 mg. a-ketoglutarate as a transaminase acceptordissolved in saline pH 7.4-7.6. After 30 minutes, rats were sacrificedby decapitation and their livers were excised, rapidly weighed, mincedin 15 ml. water and homogenized in a Potter-Elvehjem homogenizer with 5strokes of a drill press-driven Teflon pestle. Duplicate 3-m1. aliquotsof whole liver homogenates were added to tubes containing 2.1 ml. 5 NNaOH and saponified with 2.6 ml. 5 N H 804 and extracted twice with 5ml. of petroleum ether (B.P. 40-60 C.). Supernatants were added directlyto glass counting vials, evaporated to dryness and 10 ml. oftoluene-PPO-POPOP scintillation fluid was added. Samples were analyzedfor absolute activity in a Packard Tri-carb scintillation counter.Resulting data was expressed as nanomoles *C-alanine incorporated/gramof tissue/30 minutes.

INHIBITION OF IN VIVO RATES OF LIPO GENESIS BY ORAL ADMINISTRATION OFEPOXIDES 1 Lipogenesis Nanomoles C-ala- Percent Epoxide I nine/g.liver/30 min. inhibition ASV 1. 022. 45:58.7 1 (21)(:l:)-threo-epoxyaconi tic acid- 280. 6*81. 9 73 ()-threo-epoxyaconiticacid. 198. 7132. 5 (5) 81 (+)-thren-epoxyaconitic acid 364. 85:72 0 (5)64 (:I:)-erythroepoxyaconitic acid 808 6i189 8 (5) 20(-)-erythro'epoxyaconitic acid trimethyl ester 624. 55:175. 6 (5) 38 1Rats were prefasted 2 days, meal-fed the G-70 diet for 12 days andassayed in viva immediately after the completion of the last meal.

9 Den'vatives were suspended in ASV (2.63 mmoles/kg.) and given bystomach tube 60 min. before feeding.

* Mean :1: SEM for the number of rats indicated in parentheses.

INHIBITION OF IN VIVO RATE OF LIPO GENESIS BY ORAL- LY ADMINISTERED(d:)-THREO-EPOXY-ACONITIC ACID Dose admin- Lipogenesis istered (insaline solutio Nanomoles C-ala- Percent Group mmoles/kg. nine/ g.liver/30 min. inhibition 1 1, 091. 4:1:55. 6 I (31) 0 5. 26 222. 0:i:41.8 (15) 80 2. 63 372. 25:66. 7 (19) 66 1. 32 668. 73:129. 0 (9) 39 0. 66769. 2:i:52. 1 (9) 29 1 Rats were prefasted for 2 days and meal-fed theG40 diet for 9 days. On the last day of feeding they were given thederivatives 60 min. prior to the meal and assayed in vivo 5 hours afterthe initiation of feeding.

I Mean :1: SEM for the number of rats indicated in parentheses.

EFFECT OF FEE-TREATMENT WITH (i)-THREO-EPOXY ACONITIC ACID ON THE RATEOF IN VIVO LIPO GENESIS Concentra- Administration (in saline tion time(hr.

In vivo lipogenesis solution), before ieed- Nanomles C-a1a- PercentGroup mmoles/kg. ing), hours nine/g. liver/3O min. inhibition 2. 63 16and 12 616. 91:173. 5 (5) 48 Rats were preiasted 2 days and meel-ied the(i-70 diet for 10 days. They were assayed in vivo 5 hours afterinitiation of feeding.

I Derivatives were given 16 hours before feeding (day 9) and 12 hoursbefore feeding (day 10).

I Mean :1: SEM for number of rats indicated in parentheses.

14 What is claimed is: 1. A compound of the formula References CitedUNITED STATES PATENTS 2,816,125 12/1957 Allen et a1. 260456R OTHERREFERENCES Lowenstein: Chem. Abstracts, vol. (1971), p. 238.

Subject Index, Chem. Abstracts, vol. 75 (1971), p. 3185s.

JOSEPH E. EVANS, Primary Examiner US. Cl. X.R.

260-343.6, 348 A, 348.5 L, 456 P, 484 P, 535 P; 424305, 317

